No abstract

There is strong evidence for a role of prostaglandin E2 (PGE2) in cancer cell proliferation and tumor development. In PGE 2 biosynthesis, cyclooxygenases (COX-1/COX-2) convert arachidonic acid to PGH2, which can be isomerized to PGE2 by microsomal PGE-synthase-1 (MPGES-1). The human prostate cancer cell line DU145 expressed high amounts of MPGES-1 in a constitutive manner. MPGES-1 expression also was detectable in human prostate cancer tissues, where it appeared more abundant compared with benign hyperplasia. By using shRNA, we established stable and practically complete knockdown of MPGES-1, both in DU145 cells with high constitutive expression and in the non-small cell lung cancer cell line A549, where MPGES-1 is inducible. For microsomes prepared from knockdown clones, conversion of PGH 2 to PGE2 was reduced by 85-90%. This resulted in clear phenotypic changes: MPGES-1 knockdown conferred decreased clonogenic capacity and slower growth of xenograft tumors (with disintegrated tissue structure) in nude mice. For DU145 cells, MPGES-1 knockdown gave increased apoptosis in response to genotoxic stress (adriamycin), which could be rescued by exogenous PGE 2. The results suggest that MPGES-1 is an alternative therapeutic target in cancer cells expressing this enzyme.arachidonic acid ͉ cyclooxygenase ͉ eicosanoid ͉ prostaglandin E2

SummaryDicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. In addition to revealing the dual RNA and protein binding properties of Dicer C-terminus, our results may provide a link between miRNA-mediated regulation of gene expression and inflammation.

We previously showed that, in vitro, hyperforin from St. John's wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B(4) formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.

The activity of 5-LO (5-lipoxygenase), which catalyses two initial steps in the biosynthesis of pro-inflammatory LTs (leukotrienes), is strictly regulated. One recently discovered factor, CLP (coactosin-like protein), binds 5-LO and promotes LT formation. In the present paper we report that CLP also stabilizes 5-LO and prevents non-turnover inactivation of the enzyme in vitro. Mutagenesis of tryptophan residues in the 5-LO beta-sandwich showed that 5-LO-Trp102 is essential for binding to CLP, and for CLP to support 5-LO activity. In addition, the stabilizing effect also depended on binding between CLP and 5-LO. After mutations which prevent interaction (5-LO-W102A or CLP-K131A), the protective effect of CLP was absent. A calculated 5-LO-CLP docking model indicates that CLP may bind to additional residues in both domains of 5-LO, thus possibly stabilizing the 5-LO structure. To obtain further support for binding between CLP and 5-LO in a living cell, subcellular localization of CLP and 5-LO in the monocytic cell line Mono Mac 6 was determined. In these cells, 5-LO associates with a nuclear fraction only when differentiated cells are primed with phorbol ester and stimulated with ionophore. The same pattern of redistribution was found for CLP, indicating that the two proteins associate with the nucleus in a co-ordinated fashion. The results of the present study support a role for CLP as a chaperoning scaffold factor, influencing both the stability and the activity of 5-LO.